Use of a culture-independent approach to characterize aerosolized bacteria near an open-freestall dairy operation

Animal manures are known to harbor a variety of zoonotic pathogens, which are suspected of being transported off-site as aerosols from confined feeding operations. In this study, aerosols were collected using a high-volume sampler downwind from a 10,000 cow open-freestall dairy and nearby fields being sprinkler irrigated with wastewater. DNA extracts were prepared from the aerosol samples, then a region of the16S ribosomal RNA gene was sequenced for bacterial identification and phylogenetic classification. At the dairy and irrigation sites, Proteobacteria was the most abundant phylum, representing 78% and 69% of all sequences, respectively, while Actinobacteria, Bacteroidetes and Firmicutes represented only 10% or less of the sequences. Of the 191 clones sequenced from the dairy aerosol samples, 6 sequences were found to be homologous with uncultured bacteria from cow milk, rumen, and fecal samples. However, none of the sequence matches were affiliated with bacteria known to be pathogenic to otherwise healthy humans. Although our results do suggest a high diversity among the aerosolized bacteria, the sampling strategy employed in this study may not account for the variable nature of bioaerosol emissions

Airborne Endotoxin from Indoor and Outdoor Environments: Effects of Sample Dilution on the Kinetic Limulus Amebocyte Lysate (LAL) Assay.

Airborne endotoxin in occupational environments are a potential respiratory hazard to individuals. In this study, total and inhalable airborne endotoxin samples were collected via filtration from inside animal housing units and downwind from agricultural production sites and a wastewater treatment plant. Filter extracts were then diluted to examine the effect of interfering substances on the kinetic Limulus amebocyte lysate (LAL) assay. In most cases, the overall endotoxin concentration was shown to decrease with increasing dilution up to 1,000-fold, suggesting the presence of enhancing substances in the filter extracts. This dilution dependent effect was most prominent in the inhalable endotoxin samples, while total endotoxin samples displayed little effect. Using a joinpoint regression model, it was determined that a dilution factor of 50 to 100 was generally sufficient to eliminate the presence of enhancing substances. After screening the data for dilution dependent effects, the airborne endotoxin concentrations were determined. The highest endotoxin concentrations, ranging from 2,841 to 49,066 endotoxin units (EU) m-3 of air, were found inside swine farrowing and finishing barns. Airborne endotoxin concentrations were 10- to 1,000-fold lower inside a dairy barn and downwind of other agricultural production sites and a wastewater treatment plant. Examination of dilution dependent effects should be considered essential when utilizing the LAL assay, especially if values are to be used for regulatory purposes

Fate and Transport of Bioaerosols Associated with Livestock Operations and Manure

Airborne microorganisms and microbial byproducts from intensive livestock and manure management systems are a potential health risk to workers and individuals in nearby communities. This report presents information on zoonotic pathogens in animal wastes and the generation, fate, and transport of bioaerosols associated with animal feeding operations and land applied manures. Though many bioaerosol studies have been conducted at animal production facilities, few have investigated the transport of bioaerosols during the land application of animal manures. As communities in rural areas converge with land application sites, concerns over bioaerosol exposure will certainly increase. While most studies at animal operations and wastewater spray irrigation sites suggest a decreased risk of bioaerosol exposure with increasing distance from the source, many challenges remain in evaluating the health effects of aerosolized pathogens and allergens in outdoor environments. To improve our ability to understand the off-site transport and diffusion of human and livestock diseases, various dispersion models have been utilized. Most studies investigating the transport of bioaerosols during land application events have used a modified Gaussian plume model. Because of the disparity among collection and analytical techniques utilized in outdoor studies, it is often difficult to evaluate health effects associated with aerosolized pathogens and allergens. Invaluable improvements in assessing the health effects from intensive livestock practices could be made if standardized bioaerosol collection and analytical echniques, as well as the use of specific target microorganisms, were adopted

Estimation of infectious risks in residential populations exposed to airborne pathogens during center pivot irrigation of dairy wastewaters

In the western United States where dairy wastewaters are commonly land applied, there are concerns over individuals being exposed to airborne pathogens. In response, a quantitative microbial risk assessment (QMRA) was performed to estimate infectious risks after inhalation exposure of pathogens aerosolized during center pivot irrigation of diluted dairy wastewaters. The dispersion of pathogens (Campylobacter jejuni, Escherichia coli O157:H7, non-O157 E. coli, Listeria monocytogenes, and Salmonella spp.) was modeled using the atmospheric dispersion model, AERMOD. Pathogen concentrations at downwind receptors were used to calculate infectious risks during one-time (1, 8, and 24 h) and multi-day (7 d at 1 h/d) exposure events using a Beta-Poisson dose-response model. This assessment considered risk of infection in residential populations that were 1 to 10 km from a center pivot operation. In the simulations, infectious risks were estimated to be the greatest in individuals closest to the center pivot, as a result of a higher pathogen dose. Based on the results from this QMRA, it is recommended that wastewaters only be applied during daylight hours when inactivation and dilution of airborne pathogens is highest. Further refinement of the dispersion and dose-response models should be considered to increase the utility of this QMRA

The characterization of trace metals and organics in spent foundry sands over a one-year period

Millions of tons of spent sand, used to create metal casting molds, are generated by the foundry industry each year in the United States. Not surprisingly, spent foundry sands (SFSs) are an excellent substitute for virgin sands that are currently used in manufactured soils and geotechnical applications. The purpose of this study was to characterize trace metals and EPA-priority polycyclic aromatic hydrocarbons (PAHs) and phenolics in ferrous and non-ferrous SFSs over a one-year period. Overall, the total metal concentrations in the SFSs were similar to those found in native soils, while the PAHs and phenolic concentrations were relatively low. Metal leaching tests were also performed, which revealed that the SFSs have a low metal leaching potential under the specific test conditions. The data from this study suggests that the majority of SFSs are not hazardous in nature, except those that use olivine sands or are from brass foundries, due to the presence of elevated concentrations of Ni or Cu, Pb, and Zn, respectively. This information will be useful to environmental regulators who are considering including SFSs in their beneficial use regulations

Contrasting genetic diversity of listeria pathogenicity islands 3 and 4 harbored by non-pathogenic listeria spp.

Listeria monocytogenes causes the severe foodborne disease listeriosis. Several clonal groups of L. monocytogenes possess the pathogenicity islands Listeria pathogenicity island 3 (LIPI-3) and LIPI-4. Here, we investigated the prevalence and genetic diversity of LIPI-3 and LIPI-4 among 63 strains of seven non-pathogenic Listeria species from the natural environment (i.e., wildlife and surface waters). Analysis of the whole genome sequence data suggested that both islands were horizontally acquired but differed considerably in their incidence and genetic diversity. LIPI-3 was identified among half of the Listeria innocua strains in the same genomic location as Listeria monocytogenes (guaA hotspot) in a truncated form with only three strains harboring full-length LIPI-3, and a highly divergent partial LIPI-3 was observed in three Listeria seeligeri strains, outside the guaA hotspot. Premature stop codons (PMSCs) and frameshifts were frequently noted in the LIPI-3 gene encoding listeriolysin S. On the other hand, full-length LIPI-4 without any PMSCs was found in all Listeria innocua strains, in the same genomic location as L. monocytogenes and with ~85% similarity to the L. monocytogenes counterpart. Our study provides intriguing examples of genetic changes that pathogenicity islands may undergo in non-pathogenic bacterial species, potentially in response to environmental pressures that promote either maintenance or degeneration of the islands. Investigations of the roles that LIPI-3 and LIPI-4 play in non-pathogenic Listeria species are warranted to further understand the differential evolution of genetic elements in pathogenic versus non-pathogenic hosts of the same genus

Airborne endotoxin concentrations at a large open-lot dairy in southern Idaho

Endotoxins are derived from gram-negative bacteria and are a potential respiratory health risk for animals and humans. To determine the potential for endotoxin transport from a large open-lot dairy, total airborne endotoxin concentrations were determined at an upwind location (background) and five downwind locations on three separate days. The downwind locations were situated at of the edge of the lot, 200 and 1390 m downwind from the lot, and downwind from a manure composting area and wastewater holding pond. When the wind was predominantly from the west, the average endotoxin concentration at the upwind location was 24 endotoxin units (EU) m?3, whereas at the edge of the lot on the downwind side it was 259 EU m?3. At 200 and 1390 m downwind from the edge of the lot, the average endotoxin concentrations were 168 and 49 EU m?3, respectively. Average airborne endotoxin concentrations downwind from the composting site (36 EU m?3) and wastewater holding pond (89 EU m?3) and 1390 m from the edge of the lot were not significantly different from the upwind location. There were no significant correlations between ambient weather data collected and endotoxin concentrations over the experimental period. Th e downwind data show that the airborne endotoxin concentrations decreased exponentially with distance from the lot edge. Decreasing an individual’s proximity to the dairy should lower their risk of airborne endotoxin exposure and associated health effects

Recovery of culturable of Escherichia coli O157:H7 during operation of a liquid-based bioaerosol sampler

Collection fluids used in liquid-based bioaerosol samplers can influence the viability of microorganisms. In this study we determined the recovery efficiency of vegetative E. coli O157:H7 cells that were spiked into low viscosity evaporating collection fluids during operation of a BioSampler™ for up to 90 min at room temperature. The collection fluids tested were distilled (DI) water, phosphate-buffered saline (PBS) and osmoprotectants consisting of peptone and/or antifoam or betaine at 0.1% (w/w) in DI water. Using DI water, there was a rapid decline in the recovery of culturable E. coli, with only 11, 3, and 0% being recovered after 30, 60, and 90 min, respectively. Recoveries were substantially greater with use of PBS (53, 25, and 16%, respectively) but not as high as with use of the osmoprotectants. Peptone solutions, which are commonly used in liquid-based bioaerosol samplers, allowed for the recovery of 87% of the E. coli after 90 min. However, the control data indicate that some cellular growth did occur, which could be offsetting the recovery of culturable E. coli towards slightly higher values. When an antifoaming agent was added to the peptone solution there was little overall change in the amount of E. coli recovered. Betaine was also determined to be an effective osmoprotectant, with 101, 77, and 41% of E. coli recovered from the impingers at 30, 60, and 90 min, respectively. The results from this study support the incorporation of osmoprotectants in collection fluids, but not use of DI water and PBS, when BioSampler runtimes up to 90 min will be utilized. Runtimes longer than 30 min are sometimes necessary when the airborne concentration of a target organism is low and one is trying to increase the probability of detection

Qualitative and quantitative methodologies for determination of airborne microorganisms at concentrated animal-feeding operations

The generation of airborne microorganisms from concentrated animal-feeding operations (CAFOs) is a concern from a human and animal health perspective. To better understand the airborne microorganisms found in these environments, a number of collection and analytical techniques have been utilized and will be discussed in this review. The most commonly used bioaerosol collection method is the liquid impingement format, which is suitable with a number of culture-based and non-culture molecularbased approaches, such as polymerase chain reaction. However, the vast majority of airborne microorganism studies conducted at CAFOs utilize culture-based analyses. Because of the limitations often associated with culturebased analyses, we focused our discussion on the application of molecular-based techniques to identify and/or quantify microorganisms, as they have promising application in bioaerosol research. The ability to rapidly characterize airborne microorganisms will help to ensure protection of public and environmental health

Ambient Endotoxin Concentrations and Assessment of Offsite Transport at Open-Lot and Open-Freestall Dairies

Endotoxins are derived from gram-negative bacteria and are a potent inducer of inflammatory reactions in the respiratory tract when inhaled. To assess daily fluctuations of airborne endotoxin and their potential for transport from dairies, endotoxin concentrations were monitored over an 8-h period at upwind (background) and downwind (5 m from edge of dairy) locations on three separate days at two dairies. The dairies consisted of an open-lot or an open-freestall production system, both of which were stocked with 10,000 milking cows. Upwind concentrations were stable throughout the sampling period, averaging between 1.2 and 36.8 endotoxin units (EU) m-3, whereas downwind concentration averages ranged from 179 to 989 EU-3. Downwind endotoxin concentrations increased with wind speed, animal activity, and lot management practices, resulting in concentrations up to 136-fold higher than upwind concentrations. An area-source model was used to predict downwind ground-level endotoxin concentrations at distances up to 2000 m from the production facilities. Predicted concentrations decreased with distance and reached background levels within 500 to 2000 m, depending on the source emission rate and meteorological conditions